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Y. Murata
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-066 - Site-Selected Chromatin-Immunoprecipitation (ChIP) Analysis by Laser Captured Microdissection (ID 1752)
09:30 - 17:00 | Author(s): Y. Murata
- Abstract
Background:
High throughput sequencing methods such as exome sequencing, RNA sequencing, Chromatin–immunoprecipitation (ChIP) sequencing are essential tools for cancer research. However, these fine and delicate analyses contain several methodological problems. For example, although tumor mass may be suitable for mutation analysis, histological heterogeneity of the tumor tissue causes insufficient results especially for epigenetic or RNA analyses. Besides, the cancer-associated stromal cells and immune cells in the tumor will also affect the results. In this study, we tried ChIP for tiny but pure tumor samples which were selected by laser captured microdissection and verified its availability for ChIP sequence analysis.
Methods:
We used a lung adenocarcinoma frozen tissue harboring EGFR L858R mutation. After formalin fixation (1%, 10min), tumor cells, stroma cells and immune cells were microdissected separately by LMD4000 (Leica) and ChIP was performed to using H3K4me3 anti-body. Then, the quality was confirmed by real-time PCR for CCR7 which is one of the tumor specific markers and CD3 which is representative T lymphocyte marker. Sanger sequence for EGFR L858R mutation was also analyzed for confirmation that each sample was dissected and extracted correctly.
Results:
Only from the sample of tumor cells, we detected EGFR L858R mutation by Sanger sequence but from stromal cells and immune cells, we did not detect EGFR mutation. The result showed that we extracted samples correctly. And H3K4me3 mark at CCR7 gene was detected only from tumor cells and was not detected from the other samples. Moreover, H3K4me3 mark at CD3 gene was detected from stroma cells and immune cells but not tumor cells. These results indicated that microdissection method is useful and necessary method for ChIP analysis.
Conclusion:
Microdissection can be applied for epigenetic analysis like ChIP method. Our results indicated that microdissection method is useful for tumor-cell-specific epigenome profiling by ChIP sequencing.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-091 - Expression of Cytoplasmic ECT2 as a New Prognostic Marker for Early-Stage Lung Adenocarcinoma (ID 2194)
09:30 - 17:00 | Author(s): Y. Murata
- Abstract
Background:
We have examined genetic abnormalities in early-stage lung adenocarcinoma (LAd) using array-comparative genomic hybridization (array-CGH) and found that ECT2 amplification and overexpression can be a new prognostic marker (Cancer Science, 2014). In normal cells, ECT2 is localized in the nucleus, and its function is associated with cytokinesis. In cancer cells, however, ECT2 is thought to exist in the cytoplasm as well as the nucleus. In the cytoplasm, ECT2 is reported to bind to PKCi-Par6a and activate the Rac1 and MAPK pathway. Therefore, cytoplasmic ECT2 is thought to be associated with tumor growth and invasion. In the present study, we examined the clinicopathological implication of cytoplasmic ECT2 in terms of patient outcome, and also the biological significance of cytoplasmic ECT2 using lung adenocarcinoma cell lines.
Methods:
To examine the clinicopathological implication of cytoplasmic ECT2, 66 cases of various types of lung adenocarcinoma were examined using immunohistochemistry (IHC). Nine lung adenocarcinoma cell lines – A549, Calu-3, HCC827, LC-2/ad, NCI-H23, NCI-H1650, NCI-H1975, PC-9 and RERF-LC-KJ – were genetically examined for ECT2 amplification using FISH and for intracellular localization of ECT2 by Western blotting.
Results:
Overexpression of ECT2 in the nucleus was closely associated with the MIB-1 index (r=0.76) and was a strong prognostic factor of lung adenocarcinoma (OS; P=0.0096, DFS; P=0.019). On the other hand, cytoplasmic ECT2 was also associated with patient outcome (OS; P=0.02, DFS; P=0.023). Two of the nine lung adenocarcinoma cell lines, Calu-3 and A549, expressed ECT2 in the cytoplasm as well as the nucleus.Figure 1
Conclusion:
Cytoplasmic ECT2 is a prognostic factor of lung adenocarcinoma, and some lung adenocarcinoma cell lines show localization of ECT2 in the cytoplasm as well as the nucleus.
